1. Field of the Invention
The present invention is concerned with a novel microorganism and microbiological process for the production of L-threonine. L-threonine is an essential amino acid, used in various nutrient and medical compositions. Moreover, threonine is an important additive to animal fodder, a valuable reagent used in the pharmaceutical and chemical industries, and a growth factor for amino acid-producing microorganisms used in the production of, for example, lysine and homoserine.
2. Discussion of the Background
Prior to the present invention, natural strains of L-threonine-producing microorganisms and artificial mutants thereof have been used to fermentatively produce L-threonine. L-threonine-producing artificial mutants belonging to the genera Escherichia, Serratia, Brevibacterium and Corynebacterium are known, and most of them are resistant to .alpha.-amino-.beta.-hydroxyvaleric acid. With respect to the genus Escherichia, methods for producing L-threonine using a strain transformed with a recombinant plasmid DNA comprising a threonine operon are shown in Japanese Laid-Open Patent Application Nos. 55-131397, 59-31691 and 56-15696, and in PCT Laid-Open Application No. 90-04636.
Primary carbon sources added to the fermentation media of these L-threonine-producing microorganisms include glucose, sucrose, starch hydrolysate and molasses. Based on (1) the level of threonine biosynthesis and (2) the expenditure coefficient, defined as the value (in g) of sugar necessary to produce 1 g of L-threonine, E. coli strain BKIIM B-3996 (hereafter "strain B-3996" or "B-3996") is the best of the strains disclosed (PCT Laid-Open Application No. 90-04636). Strain B-3996 can synthesize up to 85 g/l threonine, with an expenditure coefficient of 2 g sugar per 1 g of threonine, provided that a sugar-ammonium mixture is added to the nutrient medium (in response to a signal from the pH sensor used in a standard laboratory fermenter during cultivation).
However, most strains used to produce L-threonine, such as E. coli BKIIM B-3996, are L-isoleucine auxotrophic. Therefore, an enzyme in the pathway from L-threonine to L-isoleucine is defective. Accordingly, L-isoleucine must be added to cultures of L-threonine-producing strains. L-Isoleucine auxotrophy in L-threonine-producing strains prevents (1) by-production of isoleucine, thus decreasing L-threonine accumulation, and (2) surplus production of L-isoleucine, thus suppressing expression of the threonine operon(s) through the corresponding attenuator.
When using such an isoleucine-dependent strain, strict control of the amount of isoleucine added into a medium is required, because growth of the strain and threonine productivity must be moderately balanced. The more isoleucine added to the medium, the more the strain grows. However, threonine productivity is simultaneously repressed in an inverse manner.
From this point of view, strain B-3996 requires isoleucine (known as "leaky-type"), and as a result, exhibits low productivity of L-threonine in media containing molasses as both a carbon source and an energy source. Molasses is a non-nutrient raw material which is much cheaper than sucrose, but which also contains a high amount of amino acids (ex. isoleucine). Thus, although it is highly desired in the art to use molasses as both a carbon source and an energy source, the isoleucine present in molasses suppresses production of L-threonine in L-threonine-producing bacteria.